In general, animal cell culture is a preferred technique in the industry for overexpressing the target protein. Because proteins with industrial value are mostly human or animal derived proteins, and specific protein modification mechanisms (glycosylation, phosphorylation, amidation) are carried out easily in animal cells. The animal cells currently used in industry are CHO (Chinese Hamster Ovary), BHK (Baby Hamster Kidney) and myeloma cells, where the target protein is expressed by transfecting expression vector into the cells, similar to the microorganism based expression system.
However, animal cells have a disadvantage of showing low level of transfected foreign gene expression compared to an expression in the microorganism. The system widely used in the industry to overcome this disadvantage is the foreign gene amplification system, which uses dihydrofolate reductase (DHFR) gene and its gene activation inhibitor, methotrexate (MTX). This system is based on the phenomenon of the DHFR gene required for the survival and the foreign gene located close by being amplified together. In detail, the gene coding for the target protein and gene coding for the selective marker, DHFR protein, that are inserted in the same region of the chromosomal DNA are amplified simultaneously when the concentration of MTX is increased artificially.
It has been previously reported that the gene located near the DHFR gene in the expression vector is amplified simultaneously when treated with MTX (Kaufman et al. Mol Cell Biol. July; 5(7):1750-9(1985)). There is a report of high level of a foreign gene being co-expressed in the animal cell when it is inserted in the vicinity of the DHFR gene in the expression vector (Alt et al. Cold Spring Hath Symp Quant Biol. 42 Pt 2:649-57(1978); U.S. Pat. No. 4,656,134).
Gene amplification is generally a very rare phenomenon, but there are indications that acquiring gene amplified cells could be achieved through selecting cells that are resistant to the serially increased MTX concentration. It takes about 3-4 weeks for the MTX resistant colonies to form, and several multiple steps of amplification process to achieve industrially significant levels of amplification using MTX concentrations ranging from 50 nM to 500 mM.
However, during the process of inducing gene amplification using MTX treatment, problems such as reduction in cell growth rate and in productivity may occur. For instance, there is a report indicating a decrease in the level of the recombinant protein expression, rather than an increase, despite the increase in MTX concentration (Kaufman et al. Mol Cell Biol. J;5(7):1750-9(1985)). Similarly, there is a case report of significantly increasing the gene amplification effect of MTX by mutating the DHFR gene control factor sequence in the expression vector (Bai et al. Zhonghua Yi Xue Za Zhi. February 25; 83 (4):333-7(2003)).
Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.